International Journal of Advances in Science and Technology, Vol. 3, No.

3, 2011

Designing Pharmacophores for COX 2 Enzyme: An In Silico Approach

Sreevatsa A.N1., Shaukath Ali.M2.,Vinitha.R Pai3, Badrunnisa.S4*.
1

Technical Support Officer. HCL, Chennai, India. sreevatsa.agrahara@gmail.com

2

Hottur steels cantonment Bellary, India. shaukath.m@gmail.com

Department of Biochemistry, Yenepoya Medical college, Yenepoya University Darelakatte Mangalore, Karnataka, India vinitharpai@gmail.com Department of Biotechnology, BITM, Bellary, Karnataka, India. badrunnisa.s@gmail.com Abstract

The current NSAID tend to show a lot of side effects when they are administered to a patient. These side effects should be reduced without disturbing the specificity of the drug, in other words the key structural features of an NSAID should be retained. Current study focuses on developing a lead structure for NSAID by comparing the structures of few best performing commercial drugs such as diclofenac, aceclofenac, aspirin and capsaicin (naturally found in chilly, known for its anti inflammatory property). The key structural features responsible for the side effects and those features contributing to specificity in these drugs were identified. The latter structural features were retained while the earlier are minimized. The lead structure so obtained was tested for its specificity by docking into the active site of the pain causing enzyme cyclooxygenase-2 and its ADME properties were checked by computational ADME TOX tool of pharma algorithms. An overlap of the docking figures of all the four reference pharmacophores on COX 2 enzyme was done to check the extent of overlap of the specificity features which could be the basis for the design of a novel ligand with minimal side effects. In conclusion, the results show that the modified drug structure clears the Lipinski rule and has minimal possible side effects. The lead compound has also retained the entire necessary pharmacophore feature and hence the specificity was not altered. Key words: Cpsaicin, ligand based drug design, Insilco ADME-TOX. Abbreviations: NSAID: Non steroidal Anti-inflammatory drug ADME TOX: Absoption, distribution, metabolism and excretion toxicity ADME: Absoption, distribution, metabolism and excretion QSAR: Quantitative structure-activity relation ship GOLPE: Generating optimal linear PLS estimations PLS Partial least squares COX-2 : Cyclo oxygenase--2 SMILES: Simplified molecular input line entry specification WLN : Wiswesser Line Notation ROSDAL: representation of organic structure description arranged linearly SLN: Single line notation RMS: root mean squared

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Introduction
Rational drug design is one of the major challenges in structural and computational biology. The concept of indirect drug design tries to circumvent these hurdles [1-4]. Computer-aided ligand design is one such active, challenging, and multidisciplinary research field that blends knowledge of biochemistry, physics, and computer sciences. Whenever it is possible to determine experimentally or to model the three-dimensional structure of a pharmacologically relevant enzyme or receptor, computational approaches [5] can be used to design specific high-affinity ligands. If the knowledge on their binding sites is absent, assuming that drugs binding in the same pocket have common properties, the binding sites can be elucidated by comparing appropriate similarity measures [6]. In indirect drug design, the analysis is based on the comparison of the stereochemical and physico-chemical features of a set of known active/inactive molecules; lead structures are designed on the basis of the pharmacophore models obtained by such analysis [7]. The final structure so formed maybe subjected to 3D QSAR tools such as GOLPE, QUANTUM, etc., to understand the structure-activity relationship. There is increasing interest in the early prediction of absoption, distribution, metabolism and excretion (ADME) properties of compounds, in order to increase the success rate of compounds reaching drug development stage [8]. The use of models to cover multiple ADME-Tox endpoints will increase the overall breadth of coverage. Computational toxicology is an emerging and innovative field composed of disciplines and methodological approaches [8] designed to understandand safeguardpublic health and the environment from harmful effects that may be caused by exposure to pollutants in the air, water, soil, and food [9]. The large numbers of chemicals that need evaluation and the various legal statutes that regulate chemicals have traditionally made it impossible for the science to evaluate every chemical with the most rigorous testing strategies. Instead, standard toxicity tests have been limited to only a small number of chemicals. Today, however, the young field of computational biology offers the possibility that, with advances in the various sub disciplines of computational biology (e.g., genomics, proteomics, and metabolonomics), and the application of the tools of computational biology, an easier assessment of the risk the chemicals pose to human health and the environment is possible available. This study is termed Computational Toxicology. In this study, three dimensional structures of four ligands namely, aspirin, aceclofenac, diclofenac and capsaicin have been docked on the three dimensional strucrture of their target protein cyclooxygenase 2 (COX 2). The four docked structures were then overlapped with one another to check the extent of overlap and to design the novel pharmacophore for COX2. In conclusion, the novel pharmacophore should have the lone benzene ring with its R group which probably plays a major role in inhibition as it is the common feature among all the drugs.

Materials and Methods

Indirect Insilco Drug Design The wet lab assay on COX-2 homolog i.e., lipoxygenase using aceclofenac, diclofenac and aspirin has given an idea on which compounds can be selected as reference to obtain final 3D pharmacophore positions. The structure of the assayed compounds was first downloaded from the Pubchem compound (Fig. 1-4)[10]. SMILES -Simplified molecular input line entry specification The simplified molecular input line entry specification or SMILES is a specification for unambiguously describing the structure of chemical molecules using short ASCII strings. SMILES strings can be imported by most molecule editor for conversion back into two dimensional drawings or three dimensional models of the molecules. The original SMILES specification was developed by Authur Weninger and David Weninger in the late 1980s. It has since been modified and extended by others, most notably by Daylight Chemical Information Systems Inc. In 2007, an open standard called "Open SMILES" was developed by the Blue Obelisk open-source chemistry community. Other 'linear' notations include the Wiswesser Line Notation (WLN), ROSDAL and SLN (tripios pdb code cx-2). In terms of a graph-based computational procedure, SMILES is a string obtained by printing the symbol nodes encountered in a depth-first tree

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International Journal of Advances in Science and Technology, Vol. 3, No.3, 2011 traversal of a chemical graph. The chemical graph is first trimmed to remove hydrogen atoms and cycles are broken to turn it into a spanning tree. Where cycles have been broken, numeric suffix labels are included to indicate the connected nodes. Parentheses are used to indicate points of branching on the tree. The generated SMILES for each of the reference pharmacophores used in this study is as follows Aceclofenac Diclofenac Aspirin Capsaicin : O=C(O)COC(=O)Cc1ccccc1Nc2c(Cl)cccc2Cl : O=C (O) Cc1ccccc1Nc2c (Cl) cccc2Cl : CC (=O) Oc1ccccc1C (=O) O : COc1cc (CNC (=O) CCCCC=CC(C) C) ccc1O

Computational ADME & Toxicity study The ADME-TOX boxes provided by pharma algorithm supports SMILES as a format for input molecule and hence the accurate SMILES for each molecule is written based on the structure of each molecule respectively. Obtained SMILES are typed in the tab given in ADME-TOX boxes displayed by pharma-algorithm. The results are based on the models already present in the database of the company and these models are obtained after extensive practical application of related chemical compound onto experimental animals such as lab rats, dogs, horses, etc. Similar procedure is done to predict toxicity by changing the home page box to TOX box of pharma-algorithm [2]. Molecular Docking Docking is a part of molecular modeling. It is a method which predicts the preferred orientation of each of the two molecules, when bound to each other to form a stable complex. Knowledge of the preferred orientation in turn may be used to predict the strength of association / or binding affinity between the molecules using scoring functions. Docking is frequently used to predict the binding orientation of small molecule drug candidates to their protein targets which in turn predict the affinity and speculate activity of small molecule. Hence docking plays an important role in the rational design of drugs [11, 12,]. Docking approaches Two approaches are particularly popular within the molecular docking community. One approach uses a matching technique that describes the protein and the ligand as complementary surfaces. The second approach simulates the actual docking process in which the ligand-protein, pair-wise interaction energies are calculated. Both approaches have significant advantages as well as some limitations [10]. The tool utilized in this study is openly accessed Docking server [13]. Docking Server is an internet service that calculates the site, geometry and energy of small molecules interacting with proteins. Ligands can be docked to appropriate target proteins of choice and their interaction analyzed in 4 easy steps. The Docking Server is offered to everyone in the field of molecular modeling from beginners to professionals. Docking Server can be used for molecular docking and thorough analysis of single ligands or for high throughput docking of sdf files. The four steps involved are: Step 1 - Ligand setup, Step 2 - Protein setup, Step 3 - Start of docking calculation Step 4 - Evaluation of results, and then return to step 1. The ligands downloaded from the pubchem database are in sdf format but the docking server doesnt support this format for free version users hence the format needs to be changed. Marvin suite comes handy during changing the format. The molecules are first opened in the marvin space and saved as .pdb format. Once registered in docking server, the home page shows the tab of docking, wherein the ligand in .pdb format is uploaded. The structure of the molecule is displayed once the molecule is completely computed by the server based on its stereochemistry. Protein setup The enzyme can be uploaded directly from the protein data bank by giving the pdb code COX 2. Later the structure is confirmed after the complete 3D positions of the amino acids are computed and displayed by the server.

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International Journal of Advances in Science and Technology, Vol. 3, No.3, 2011 Start of docking calculation The user interface of docking server is very simple and is same as operating mail servers. The tab my docking initiates docking procedure, where the user is allowed to select the ligand (capsaicin / aspirin / aceclofenac / diclofenac) and also the protein (5COX - COX 2). The server takes about 10 computations at a time on one ligand to complete a docking and displays the results (Fig. 6). Evaluation of Results During molecular docking it is always found that the compound having the least total intermolecular energy of docking is the best fit compound and is taken as the best ligand for the protein (Fig. 7). Docking of diclofenac on COX 2 and Pharmacophore modeling A pharmacophore is defined as an ensemble of universal chemical features that characterize a specific mode of action of a ligand in the active site of the macromolecule in 3D space. Chemical features such as hydrogen bonds, charge interactions, hydrophobic areas (Fig. 8) maybe predicted. Pharmacophores represent chemical functions, valid not only for the currently bound, but also unknown molecules. Due to its simplicity, this method is computationally very efficient and is subsequently exceptionally well suited for virtual screening of large compound libraries. Pharmacophore modeling is universal, comprehensive and editable: Selectivity-tuning by adding or omitting feature constraints provides a plenitude of manipulation options (Table 1). LigandScout is a powerful structure-based pharmacophore generator based on a sophisticated and customizable ligand-macromolecule complex interpretation algorithm. LigandScout extracts and interprets ligands and their macromolecular environment from PDB files and automatically creates and visualizes advanced 3D pharmacophore models supporting multiple features per heavy atom to broaden the scope of a single model. A wide range of powerful editing tools generate customized, highly specific pharmacophores within a few seconds. Excluded volume recognition as well as excluded volume coats drastically increase selectivity by considering sterical characteristics of the binding site. Moreover, LigandScout supports the generation of pharmacophore models based on molecules injected into the binding site and supplies a library view for the investigation of docking poses [14]. LigandScout provides pharmacophore features such as hydrogen bond, hydrophobic interactions and ionizable areas for automated pharmacophore generation. While hydrogen bond features are defined by direction and distance constraints, hydrophobic interactions and ionizable areas have a distance constraint only. LigandScout can also generate pharmacophore models that are solely based on one or several ligands. You can create a ligand-based pharmacophore model by using the Create Pharmacophore command in the context menu of the Alignment View. For generation of a pharmacophore based on several ligands, the ligands have to be aligned before the pharmacophore generation. Selected target specific ligands are considered as reference molecules during the pharmacophore elucidation. A single alignment is based on matched chemical features (feature pairs) where one feature - from the reference element and that of the element to be aligned are used. For pharmacophores, these chemical features are given explicitely. However, for molecules these features have to be derived during the alignment algorithm and for the sake of clarity these are not visible to the user. All elements which will be selected in the alignment list will be aligned to a reference element. This reference element can be set by the user or will be chosen automatically. To perform an alignment, two or more elements are selected in the alignment list. After clicking onto the icon Align Selected Elements LigandScout will calculate one or several alignments and present them to the user (Fig. 8). The activated slider indicates that there are several alignments available. For each alignment the associated RMS (root mean squared) value of the valid matched feature deviances is shown as well as the number of valid matched features in brackets. There are cases where LigandScout will provide just a single alignment [14, 7]: 1. Three or more element will be aligned to each other 2. A perfect alignment was found (usually a molecule to its own pharmacophore, or aligning two instances of the same structure). Ligand Design: This can be achieved by the knowledge obtained by merging the pharmacophores of the best COX2 inhibiting commercial drugs such as acelofenac, diclofenac, aspirin, etc. The highest

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International Journal of Advances in Science and Technology, Vol. 3, No.3, 2011 inhibiting ability with lowest intermolecular energy of docking and least adverse health effects as predicted by the computational ADME-TOX of pharma-algorith have to be considered for the design of the novel ligand / pharmacophore. Novel ligand: The main aim of ligand design is to improve the binding ability of ligand which has been obtained by slight modification in the structure of capsaicin without causing more side effects. This can be obtained by merging pharmacophores of aceclofenac and diclofinac with capsaicin, which have proven good inhibitors in the assay. The final 3D pharmacophore positions obtained by LIGAND SCOUT were computed like jig saw to obtain final ligand structure. The structure of the compound is more optimised by reducing the total energy of the compound itself to the least. This allows the conformation to remain stable in it native environment. Ligandscout also gives an option to reduce the energy to the local minimum. The reduction was carried out to energy of 29.57 k cal.

RESULT
The Structure and properties of the reference ligands / pharmacophores of OX 2 obtained from the website were as follows (Fig. 1-4) Diclofenac (Fig. 1 and Table I). A non-steroidal anti-inflammatory agent (NSAID) with antipyretic and analgesic actions. It is primarily available as the sodium salt. Aceclofenac (Fig. 2 and Table II) Is a non-steroidal anti-inflammatory agent. In addition to anti-inflammatory actions, they have analgesic, antipyretic, and platelet-inhibitory actions. They act by blocking the synthesis of prostaglandins by inhibiting COX 2, which converts arachidonic acid to cyclic endoperoxide precursors of prostaglandins. Inhibition of prostaglandin synthesis accounts for their analgesic, antipyretic, and platelet-inhibitory actions; other mechanisms may however contribute to their antiinflammatory effects. Aspirin (Fig. 3 and Table III) The prototypical analgesic used in the treatment of mild to moderate pain. It has anti-inflammatory and antipyretic properties and acts as an inhibitor of COX 2, which results in the inhibition of the biosynthesis of prostaglandins. Aspirin also inhibits platelet aggregation and is used in the prevention of arterial and venous thrombosis. Capsaicin (Fig. 4 and Table IV) Capsaicin(8-methyl-N-vanillyl-6-nonenamide, (CH3)2CHCH=CH(CH2)4CONHCH2C6H3-4-(OH)-3(OCH3)) is the active component of chili peppers, which are plants belonging to the genus Capsicum. Carcinogenic, co-carcinogenic, anti-carcinogenic, anti-tumorigenic, tumor promotion and anti-tumor promotion effects of capsaicin have been reported in animal studies [15]. In 1997, a research team led by David Julius of UCSF showed that capsaicin selectively binds to a protein known as TRPV1 that resides on the membranes of pain and heat sensing neurons [16]. Insilico ADME Results: The in silico ADME results show that the novel ligand designed has very equivalant ADME properties as the other reference drugs tested (Table V) and are indicating that the designed novel compound has better features than its derivatives. The results distinctly show a similarity of the novel ligand with the safe features of the aceclofenac (which is the best among the reference pharmacophores used in this study) (Fig. 5 green-yellow parts) with minimal overlap on the toxic features (Fig. 5. red part) Insilco toxicity result: (Table VI) The toxicity results show that aceclofenac is the best resulting drug because it is showing least possible side effects on organs and least genotoxicity. A toxicity result of the novel compound is the most promising one and shows much better results than aceclofenac. It has the least probability of positive Ames test and also least probable side effects on different organs. Molecular docking: (Table VII) The results of molecular docking show that aceclofenac has the best binding ability and the novel ligand designed has binding ability improved from its native capsaicin structure without its native side. Results of LigandScout: Fig. 6 represents the 3D structure of a pharmacophore as shown by the use of the ligandscout software. Ligand Design: This merging studies clearly shows that the benzene ring with the R group attached to it is the major pharmacophore which is most common among all the reference pharmacophores (Fig. 7). An

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International Journal of Advances in Science and Technology, Vol. 3, No.3, 2011 optimization of the structure of capsaicin, keeping the important pharmacophores at correct position, was done for the final ligand structure. The result shows that aceclofenac has lowest intermolecular energy of docking (Table VIII) and hence most of the pharmocophoric features of the new ligand should be derived from the aceclofenac pharmacophore. Merging the pharmacophore shows that the lone benzene ring with its R group plays a major role in inhibition as it is the common feature among all the drugs (Fig. 7 a, b) and hence we must ensure that this pharmacophore is retained. Here you can see the aligned and merged pharmacophores of all the compound selected that is aspirin, diclofenac, aceclofenac and capsaicin. The pharmacophore along with the structure are merging, this mergence clearly shows that the benzene ring is the major pharmacophore which is most common among all the species and so are the R groups attached to it. On optimization of the structure of capsaicin (which has least side effects) and by keeping the important pharmacophores at correct position, the novel lignd was proposed (Fig.8). Discussion. Margo McCaffery in 1968 defined pain and considered it as a subject in medicine, pain can be caused by the formation of biological mediators called prostanoides, including prostaglandins, prostacyclin and thromboxane which is catalysed by COX enzyme (EC 1.14.99.1)[17]. Pharmacological inhibition of COX enzyme can achieve by using NSAIDs such as aspirin, diclofinac, acelofinac and ibuprofen to over come the symptoms of inflammation and pain [18]. Computational approaches help pharmaceutical field/study for modeling 3-dimensional structures of an enzyme or receptor without consuming much time. Indirect drug design is a major challenge for the structural biologist and biochemist to design specific high affinity ligands by comparing stereochemical and physicochemical features of active/ inactive molecules [7, 11]. This can be achieved by increasing the interest in the prediction of ADME properties of compounds [8]. Present study incorporates docking of three dimensional structures of four ligands (aspirin, aceclofinac, diclofinac and capsaicin) on the target protein COX-2. Currently three COX isoenzymes are known: COX-1, COX-2 and COX-3. Side effects such as peptic ulceration and dyspepsia have been reported by the present COX inhibitors [19]. Capsaicin a secondary metabolite in chili and peppers [15] is responsible for its piquancy. -hydroxy--aryl-alkanoic acids class were studied as a potential COX-2 inhibitors by molecular docking [20]. ADME-Tox profile of compounds have been used to propose the new pharmacophore[21]. Similar studies were performed on 114 analogues of 1, 2 diarylimidazole to optimize COX 2 antiinflamatory activities [22]. Present study utilizes capsaicin, a potent inhibitor for COX-2. Previous studies on capsaicin indicates that it is a neuropeptide releasing agent although the exact mechanism is still unclear [23]. Assumptions were made for simulations to remain feasible such as rigidity of receptor, conformational flexibility and protein-ligand interaction, etc [24]. This study an indirect method of drug design to propose a novel pharmacophore for COX-2. SMILES was used to generate four reference pharmacophores, namely diclofinac, aceclofinac, aspirin and capsaicin , predict their molecular structures and chemical features (fig-1-4). In silico ADME results of Capsaicin clearly differentiates and indicates the toxic features with red color, yellow less toxic and green as safe features (fig-5). It has least probability of positive Ames test and also least possible side effects on organs and least genotoxicity as shown in the (table VI). Docking is frequently used to predict the binding orientation of small molecule drug candidates to their protein targets which in turn predict the affinity and speculate activity of small molecule (Table VII) and the study shows the best binding ability. Obtained results have been used to design a pharmacophore represented in (fig-6). Ligand design carried out by merging the benzene ring with the R groups attached which is common for all the reference pharmacophores as depicted in (fig-7), optimization of final ligand structure was done by keeping the important pharmacophores at correct positions. Fig-7 a, b represent clearly that novel ligand binding at the safe features of COX-2 with total intermolecular energy of about -5.57 k.cal which implies the reduction of the side effects. More research on this particular drug may give a ligand which has much accuracy and can be used in treatment of arthritis and other chronic problems. Conclusion Ligand based drug design offers hew horizons to not only design of new drug in much faster way but also a scope to improve the current available drug. Due to the advancements done in the field of computational biology, softwares (bio wares) which work in much faster pace and yield more reliable

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International Journal of Advances in Science and Technology, Vol. 3, No.3, 2011 result are available. Computational ADME-TOX results can be used to streamline the process of drug safety testing and reducing the time of drug safety assessment. Such and other tools are to be developed by computational biologists which reduce time of bringing new drugs into market. In conclusion, computational biology appears to have tremendous application in drug design industry with the advent of computational tools which are more accurate and reliable. Table 1. Chemical features of Diclofenac Compound ID 3033 Molecular Weight 296.14864 [g/mol] Molecular Formula C14H11Cl2NO2 Table 2. Chemical features of Aceclofenac Compound ID Molecular Weight Molecular Formula XLogP3 H-Bond Donor H-Bond Acceptor 71771 354.18472 [g/mol] C16H13Cl2NO4 4.3 2 5

XLogP3 4.4 H-Bond Donor 2 H-Bond Acceptor 3 Table 3. Chemical features of aspirin Compound ID 2244 Molecular Weight 180.15742 [g/mol] Molecular Formula C9H8O4 XLogP3 1.2 H-Bond Donor 1 H-Bond Acceptor 4

Table 4. Chemical features of capsaicin Compound ID 1548943 Molecular Weight 305.41188 [g/mol] Molecular Formula C18H27NO3 XLogP3-AA 3.6 H-Bond Donor 2 H-Bond Acceptor 3

Table 5. ADME values of all the compounds for comparative study. Aceclofenac Oral bioavailability less than 30% Maximum passive absorption: 100% High solubility (LogS) At pH = 8.0 (Colon): -0.99 Diclofenac Oral bioavailability between 30% & 70% Maximum passive absorption: 100% High solubility (LogS) At pH = 8.0 (Colon): -0.47 Aspirin Oral bioavailability between 30% & 70% Maximum passive absorption: 32% High solubility (LogS) At pH = 8.0 (Colon): 0.71 Capsaicin Oral Bioavailability more than 70% Maximum passive absorption: 100% High solubility (Log S) From pH 1 to 8= -3.41 Novel compound Oral bioavailability between 30% & 70% Maximum passive absorption: 100% High solubility(LogS) At pH = 8.0 (Colon): -0.46

Table 6. Insilco toxicity on different organs. Aceclofenac Probability of positive Ames test: 0.007 Probability of effect on Blood Cardiovas cular system Gastrointe 0.06 0.84 0.90 Diclofenac Probability of positive Ames test: 0.005 Probability of effect on: Blood Cardiovas cular system Gastroint 0.22 0.88 0.75 Aspirin Probability of positive Ames test: 0.102 Probability of effect on: Blood Cardiovas cular system Gastrointe 0.07 0.30 0.17 Capsaicin Probability of positive Ames test: 0.161 Probability of effect on: Blood Cardiovasc ular system Gastrointe 0.38 0.32 0.07 Novel compound Probability of positive Ames test: 0.018 Probability of effect on: Blood Cardiovas cular system Gastrointe 0.08 0.05 0.05

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International Journal of Advances in Science and Technology, Vol. 3, No.3, 2011 stinal system Kidney Liver Lungs 0.40 0.26 0.52 estinal system Kidney Liver Lungs 0.48 0.18 0.63 stinal system Kidney Liver Lungs 0.03 0.02 0.08 stinal system Kidney Liver Lungs 0.12 0.22 0.75 stinal system Kidney Liver Lungs 0.05 0.05 0.22

LD 50 (Rat) Intraperitonial470mg/kg Oral-1600mg/kg

LD 50 (Rat) Intraperitonial210mg/kg Oral-550mg/kg

LD 50 (Rat) Intraperitonial560mg/kg Oral-2300mg/kg

LD 50 (Rat) Intraperitonial580mg/kg Oral-1600mg/kg

LD 50 (Rat) Intraperitonial84mg/kg Oral-1400mg/kg

Table 7. Docking result obtained by docking server. Compound Total intermolecular energy Aceclofenac -7.53 Diclofenac -3.5 Aspirin -4.26 Capsaicin -5.57 Novel ligand -5.70 Table 8. Alignment of different conformations of same structure in docking server, with the lowest energy consuming conformation at first and following it are higher energy consuming conformations.

Figure 1. Molecular structure of Diclofenac

Figure 2. Molecular structure of Aceclofenac

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Figure 3. Molecular structure of Aspirin

Figure 4. Molecular structure of capsaicin

Figure 5. Toxcicity features of aceclofenac RED-indicates the toxic feature,YELLOW-less toxic, GREEN-safe feature

Figure 6. Pharmacophoric features of all the compounds merged

Figure 6. Snapshot of ligand scout showing molecules merged with all structures in stick model

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Figure 7. Representation of different pharmacophores of a compound by ligand scout

Figure 8. Proposed Novel pharmacophore

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International Journal of Advances in Science and Technology, Vol. 3, No.3, 2011 References: 1. Lanas A, Scheiman J. Low-dose aspirin and upper gastrointestinal damage: epidemiology, prevention and treatment. Curr Med Res Opin. Jan;23(1):163-73, 2007. 2. www.pharma-algorithm.com 3. Maria A. Miteva, Stephanie Violas, Matthieu Montes, David Gomez1, Pierre Tuffery1 and Bruno O. Villoutreix* computational ADME TOX tool Oxford Journals Volume 34, Web Server issue Pp. W738-W744 4. Madsen, Ulf; Krogsgaard-Larsen, Povl; Liljefors, Tommy, Textbook of DrugDesign and Discovery. Washington, DC: Taylor & Francis. ISBN 0-415-28288-8, 2002. 5. Patricia H. Reggio1, Computational Methods in Drug Design: Modeling G Protein-Coupled Receptor Monomers, Dimers, and Oligomers, AAPS Journal, 8 (2) pp.E322-E33, 2006. 6. Alok Juneja, Henning Riedesel, Milan Hodoscek and E. W. Knapp* Institute of Chemistry & Biochemistry, Freie University Berlin, Fabeckstr. 36a, D-14195 Berlin, Germany, and National Institute of Chemistry, Hajdrihova 19, SI-1001 Ljubljana, Slovenia J. Chem. Theory Comput., 5 (3), pp 659673 2009. 7. Cohen, N. Claude. Guidebook on Molecular Modeling in Drug Design. Boston: Academic Press. ISBN 012178245, 1996. 8. D.S Samiulla1, V. V. Vaidyanathan1, P. C. Arun1, G. Balan1, M. Blaze1, S Bondre1, Chandrasekhar1, A. Gadakh1, R. Kumar1, G. Kharvi1, H.-O. Kim2, S. Kumar1, 1 1 1 1 2 J. A. Malikayil , M. Moger , M. K. Mone , P Nagarjuna , C. Ogbu , D. Pendhalkar1, A. V. S. Raja Rao1, G. Venkateshwar Rao1, V. K. Sarma1, S. Shaik1, G. V. R. Sharma1, S. Singh1, C. Sreedhar1, R. Sonawane1, U. Timmanna1 and L. W. Hardy,2 Rational selection of structurally diverse natural product scaffolds with favorable ADME properties for drug discovery, Molecular Diversity Volume 9, Numbers 1-3 , pp. 131-139, 2005. 9. Florian Nigsch , NJ Maximilan Macaluso , John BO Mitchell & Donatas Zmuidinavicius , Computational toxicology: an overview of the sources of data and of modelling methods, JExpert opinion on Drug metabolism and Toxicology, Vol. 5, No. 1, Pages 1-14 2009. 10. Gregory M Banik, Bio-Rad Laboratories, Philadelphia, PA, USA. Studied on Insilico, or virtual ADME-Tox prediction, 2004. 11. Butler, T, Protein-Ligand Docking: A critical Review of Molecular Dynamics. Science 21 Aug Vol. 257. no. 5073, pp. 1078 1082, 1996 12. O. Funk, E. M. Krovat, M. Fritz, T. Langer, Pharmacophore Modeling of Thrombin ActiveSite Inhibitors Institut of Pharmacy, Department Pharmaceutical Chemistry, University of Innsbruck, Innrain 52a, 6020 Innsbruck pp- 120-121, 2001. 13. Thierry Langer1, and Gerhard Wolber2 Virtual combinatorial chemistry and in silico screening: Efficient tools for lead structure discovery, J. Pure Appl. Chem., Vol. 76, No. 5, pp. 991996, 2004. 14. www.dockingsever.com 15. Introduction to Ligand scout [Online] / auth. Ligand scout // Inte:ligand. - Inte:ligand, 11 2007. - May 2009. - www.inteligand.com. 16. Johnson, Wilbur. "Final report on the safety assessment of capsicum annuum extract, capsicum annuum fruit extract, capsicum annuum resin, capsicum annuum fruit powder, capsicum frutescens fruit, capsicum frutescens fruit extract, capsicum frutescens resin, and capsaicin". Int. J. Toxicol. 26 Suppl 1: 3106, 2007. 17. Chandrasekharan NV, Dai H, Roos KL, Evanson NK, Tomsik J, Elton TS, Simmons DL, "COX-3, a cyclooxygenase-1 variant inhibited by acetaminophen and other analgesic/antipyretic drugs: cloning, structure, and expression". Proc. Natl. Acad. Sci. U.S.A. 99 (21): 1392631, 2002. 18. Green, Ga. "Understanding NSAIDs: from aspirin to COX-2". Clinical cornerstone 3 (5): 50 60, 2001. 19. Caterina MJ, Schumacher MA, Tominaga M, Rosen TA, Levine JD, Julius D "The capsaicin receptor: a heat-activated ion channel in the pain pathway". Nature 389 (6653): 81624, October 1997.

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International Journal of Advances in Science and Technology, Vol. 3, No.3, 2011 20. Sanda P. Dilber 1, Silva Lj. Dobric 2, Zorica D. Juranic 3, Bojan D. Markovic , Sote M. Vladimirov 1 and Ivan O. Juranic 4,* Docking Studies and Anti -inflammatory Activity of Hydroxy--arylpropanoic Acids Molecules, 13, 603-615, 2008. Gregory M Banik, Bio-Rad Laboratories, Philadelphia, PA, US, Studied on Insilico, or virtual ADME-Tox prediction, 2004. Desiraju, B. Gopalakrishnan, R. K. R. Jetti, A. Nagaraju, D. Raveendra, J. A. R .Sarma, M. E. Sobhia and R. Thilagavathi.Computer aided design of selective COX-2 inhibitors: Comparative Molecular Field Analysis (CoMFA), Comparative Molecular Similarity Indices Analysis (CoMSIA) and Docking studies of some 1,2-diarylimidazole derivatives G. R. J. Med. Chem, 45, 4847-4857, 2002. Ching-Wen Chen,2. T.-M.-M. Signal transduction for inhibition of inducible nitric oxide synthase and Cyclooxygenase-2 induction by capsaicin and related analogs in macrophages. British journal of pharmacology, 2003.

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Dr. Vinitha Ramanath Pai received her Ph.D in the the year 2003 from University of Mysore. This author is a life member of Association of Clinical Biochemists of India. She is currently working as an Associate Professor in the Department of Biochemistry, Yenepoya Medical College, Yenepoya University. Dr. Pai has been teaching for the past 23 years in all faculties of Health Science. Her major research interests involve Protein Chemistry, Molecular Biology & Bioinformatics. She has successfully completed a DBT funded Post Doctoral Fellowship in the Department of Biochemistry, Indian Institute of Sciences, Bangalore.

Mrs. Badrunnisa.S, persuing Ph.D from Yenepoya University. Her major research
interest involves Biochemistry & Bioinformatics. She is currently working as an Assistant Professor in the Department of Biotechnology at Ballari Institute of Technology and Management since 2003. This author is a life member of ISTE and a College Champion for Mission-10X from Wipro. She has awarded Cambridge Certificate for Best Technical Teacher from Cambridge University.

Mr. Shaukath Ali.M is presently serving for Hottur Steels as a R&D Head. This author has 06 years of teaching experience in the field of Biotechnology. He was also served as a faculty of Jazan University for a year. His research area involves Biochemistry & Nanotechnology.

Mr. Sreevatsa Agrahara has served HCL as Technical Support Officer for a period of one year. Presently he is a student of M.Tech in PESET College of Engineering, Bangalore. This authors research interest involve in Bioinformatics.

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